Project Summary Understanding the life cycle of a pathogen within different natural environments is a poorly evolved area of infectious disease research. Tick-borne relapsing fever (RF) spirochetes are one such pathogen that have adapted to colonize arthropods and mammalian blood. When a tick imbibes an infectious bloodmeal, spi- rochetes enter the midgut. Within ~ 2 weeks a population subsequently migrates and colonizes the salivary glands. However, the molecular events facilitating vector colonization and subsequent transmission to the mammalian host remain unclear. Consequently, delineating the intricacies of the life cycle of RF spirochetes is particularly relevant to public health. Limitations in RF spirochete genomics, transcriptomics, and genetics have been addressed by develop- ing the Borrelia turicatae-Ornithodoros turicata model. Utilizing a functional genomic approach, two important observations were made that this proposal builds upon: 1) after 220 generations, B. turicatae losses genes es- sential in vector colonization and/or establishing mammalian infection; 2) the 150 kb linear megaplasmid (lp150) is likely important for vector colonization and transmission. A 36 kb locus toward the 3' end of the plas- mid contains a series of genes that coded for surface proteins that are up-regulated in the tick vector compared to infected mammalian blood. The central hypothesis of this application is that there are subsets of B. turicatae genes essential in midgut and salivary gland colonization and establishing early mammalian infection. The fol- lowing aims are purposed: Aim 1: Identify genes that are essential in the tick-mammalian transmission cycle by comparative ge- nomics, and define temporal expression and protein production of B. turicatae genes localized in the 36 kb locus. The objective is to quantify the expression of genes in the 36 kb locus during the pathogens life cycle in the tick vector, and coincide the findings with protein production. Also, a comparative genomic analysis between a wild type spirochetes and noncolonizing/noninfectious strains will identify likely essential genes. Aim 2: Identify and characterize genes essential in vector colonization and transmission by mutagene- sis, imaging, and structural and biophysical analyses. The objective is to generate and evaluate B. tu- ricatae mutants, to identify defects in tick-mediated transmission to mice. The significance of these genes in the tick-mammalian transmission cycle will be further verified by site directed mutagenesis and structural and biophysical analyses. With the surface proteome of RF spirochetes serving essential roles in pathogenesis, these findings will guide strategies to interrupt the pathogen's life cycle in the tick and mammal. The results will be broadly appli- cable to Old and New World species, and address NIAID's mission to better understand and prevent disease.